We have isolated a neuron survival factor from the developing neocortex of rats and from medium conditioned by a human cell line. This factor migrates as a 200kD band in both reduced and unreduced SDS gels. The 200kD factor is distributed in regions of high glutamate binding in the developing brain and protects axotomized neurons from degeneration in vitro. Initial amino acid sequences obtained from the 200kD band reveal sequences identical to gamma or beta actin. Biochemical characterization indicates that this protein is resistant to chemical reduction with beta- mercaptoethanol and continues to migrate at 200kD on SDS gels, indicating that the beta-actin segments are covalently associated in an apparent 200kD complex. A monoclonal antibody to the 200kD factor recognizes non- actin parts of the molecule but not any well known neurotrophic factors like NGF, CNTF, and acidic or basic FGF. This is a proposal to test the hypothesis that the 200 kD species is a cytoskeletally-associated regulatory molecule, comprised of actin covalently associated with non- actin sequences, that are also neurotrophic. Furthermore, we will examine the possibility that the factor prevents cell death and promotes cell growth by regulating neuronal responses to excitatory amino acids. The specific experiments proposed are designed to obtain additional amino acid sequences (specifically non-actin sequences) of the 200kD factor in order to more fully define this neurotrophic protein and provide the basis for future experiments involving gene cloning. To accomplish this characterization, the factor will be digested and non-actin peptides will be identified by high resolution peptide analysis. Along with these biochemical experiments, we will continue to test the neuron survival promoting properties of this factor using neonatal thalamic neurons in an in vitro bioassay. In particular, we will follow up preliminary observations that suggest the factor promotes neuron survival by acting at glutamate receptors including NMDA. In vitro studies of neonatal anterior thalamic neurons will test this hypothesis specifically. The neurotoxicity of NMDA and non NMDA glutamate receptor agonists will be investigated and the effects of the 200kD factor on this toxicity (and associated changes in intracellular calcium) will be determined.